1. Field of Invention
This invention relates to novel compositions and methods for isolating and purifying assay-ready nucleic acids, particularly DNA, RNA, and enriching for mRNA from biological tissues and cells. The isolated and enriched mRNA is suitable, without further manipulation, for Northern blot and PCR (PCR=polymerase chain reaction). In particular, the present invention comprises the use of a combination of chaotropic agents, aprotic solvents, and aromatic alcohols which work synergistically to result in improved purification and yield of nucleic acids in a relatively short period of time as compared to conventional methods of nucleic acid isolation and purification. The inventive nucleic acid compositions are also excellent tissue solubilization and protein denaturing agents for general use in the recovery and analysis of radioactive substrates and vital stains (e.g. .sup.3 H tritiated lysine and trypan blue) as well in the preparation of samples for liquid scintillation counting. The inventive nucleic acid isolation methods are further particularly useful in isolating nucleic acids from plants, which are typically difficult to extract.
2. Description of Related Art
With the increase demand for RNA and DNA for use in various molecular biological studies, there has come a need for a more efficient, more rapid, and clinically useful method for isolating these nucleic acids from biological tissues and cells. Several methods and compositions have been published relating to the isolation and purification of nucleic acids from a wide array of biological samples, including plant and bacterial cells, insect cells, as well as mammalian tissue. Most of these methods, however, are timeconsuming, require expensive centrifugation equipment, and require multiple manipulative steps which tend to decrease recovery yields.
One of the earlier methods for isolating RNA from biological tissue and cell samples was reported by Cox (Methods in Enzymology, 129B: 120-129 (1968)) and is directed to the use of guanidinium thiocyanate and guanidinium chloride as protein denaturants. The Cox method involves the addition of 6M guanidinium thiocyanate to ribosomes. The RNA is then precipitated by adding ethanol to the solution, followed by centrifugation to recover the RNA. This procedure takes two days to complete and requires relatively large concentrations of guanidinium salts.
A popular method of RNA isolation is disclosed in Chirgwin, et al. (Biochim., 18: 5294-5299 (1979). In that procedure, tissue containing RNA is homogenized in a solution containing guanidinium thiocyanate, sodium citrate, and 2-mercaptoethanol. The homogenate is then centrifuged, and the supernatant is decanted and mixed with acetic acid to lower the pH to 5. The RNA is precipitated upon the addition of ethanol and is recovered in pellet form by centrifugation. A modification of this procedure involves separating the RNA from the homogenate by ultracentrifugation through a cesium chloride gradient. This method, however, requires expensive centrifugation equipment, skilled technicians, and up to two days to complete. Consequently, only a very limited number of samples can be processed simultaneously.
Feramisco, et al. (Molecular Cloning, 194-195, Cold Springs Harbor Laboratory, Cold Springs Harbor, N.Y.) reports another RNA isolation procedure wherein RNA-containing samples are homogenized in a solution of 4M guanidinium thiocyanate, 20% sodium lauryl sarcosinate (SIGMA), and 2-mercaptoethanol. Following homogenization, an equal volume of heated phenol (60.degree. C.) and sodium acetate pH 5.2 are added to the homogenate. Next, an equal amount of chloroform is added, and the mixture is cooled and centrifuged. The aqueous phase is decanted and reextracted several times with a phenol/chloroform solution in order to maximize the yield. The procedure requires a skilled technician and takes four to five hours to complete.
The current state of the art procedure for RNA isolation is disclosed in U.S. Pat. No. 4,843,155 to Chomczynski. This procedure utilizes a single monophasic extraction reagent consisting essentially of 2-5M of a guanidinium salt or acid, 40-60% phenol, optionally 2-mercaptoethanol as an antioxidant, and a sufficient amount of sodium acetate buffer to maintain the reagent at pH 4. Chomczynski discloses that a high yield of high quality RNA can be obtained by this procedure in three hours. The procedure involves homogenizing biological samples in the reagent described above, and then adding 10% chloroform to the homogenate. The resulting suspension is next centrifuged to effect separation of the suspension into an aqueous phase, organic phase, and interphase. The RNA remains concentrated in the upper aqueous phase while DNA, proteins, and cellular debris remain in the lower organic phase and interphase. The aqueous phase is decanted, and an equal portion of absolute isopropanol is added to precipitate the RNA. The precipitated RNA is subsequently centrifuged at 12,000 g to form a pellet. The supernatant is then decanted, and the resulting pellet is washed with 70% ethanol, recentrifuged, and allowed to dry. This reference expressly states that a significantly lower degree of RNA isolation will result if the pH of the reagent is not maintained at pH 4, and the concentration of water-insoluble organic solvent used to effect phase separation is not maintained at about 10% v/v.
The Chomczynski RNA isolation procedure described above, however, has several disadvantages, including the co-isolation of polysaccharides, which have the same absorbance as RNA, thus leading to inaccurate and false quantitation of RNA, as discussed in Puissant, C. and Houdebine, L., "An Improvement of the Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate/Phenol/Chloroform Extraction," Biotechniques, 8:148-149 (1991). Further, Puissant and Houdebine maintain that the procedure requires relatively high concentrations of guanidinium salts (i.e. 2-4M) and phenol (40-60%), which have been shown to interfere with subsequent oligo-dT-cellulose chromatographic polyadenylated mRNA purification, for example. Additionally, the high concentration of phenol is undesirable due to its high toxicity.
As for RNA, several methods and procedures for the isolation of DNA from biological tissue/cell samples have been published. One of the earlier methods of isolating DNA was reported by Jones (Biochim. Biophys. Acta, 10: 607-612) and involves the use of alkyltrimethylammonium bromides to precipitate DNA. This procedure, however, takes one to two days to complete.
One of the classic procedures for isolating high molecular weight DNA was reported by Marmur (Journal of Molecular Biology, 3:208-218 (1961)). The method includes up to fourteen steps which are tedious and require a certain level of expertise to perform. For example, the method includes total nucleic acid precipitation, dissolution of nucleic acids, deproteinization, reprecipitation, RNase treatment to destroy unwanted RNA, a second deproteinization, reprecipitation, dissolution of DNA, and washing steps. The process generally requires one to two days, and the best expected yield of DNA is not greater than 50%. Marmur reports that the method requires tailoring in order to obtain efficient isolation of DNA from a wide variety of microorganisms.
Another procedure for isolating DNA was also described by Marmur. The procedure has fewer steps but requires cesium chloride high speed centrifugation for up to three days.
Gross-Bellard et al., European J. Biochem., 36:32-38 (1973) is directed to a method for isolating DNA from mammalian cells. In this procedure, cells are lysed with the detergent sodium dodecyl sulfate (SDS) and Proteinase K and then deproteinized with phenol:chloroform extraction.
A procedure for isolating RNA and DNA is described in U.S. Pat. No. 4,935,352 to Seligson et al., and is directed to the isolation of both nucleic acids from biological samples using an anion exchange resin. The isolation procedure requires first adding an agent comprising sodium lauryl sulfate (SDS) and Proteinase K, in conjunction with a detergent, to lyse the cells.
Many current DNA isolation methods continue to utilize variations of the twenty-three-year old method of Marmur, which involves many tedious time and labor intensive manipulations involving the use of hazardous phenol solvents. Consequently, there is a need for a rapid, simple, efficient, and safe method of isolating high molecular weight DNA from biological tissues/cells.